Project Summary/Abstract The objective of this proposal is to characterize the cancer-specific alteration of a metabolic complex of human glucose metabolism and its functional contribution to cancer cell metabolism. Recent advances in our knowledge of the spatial assemblies of metabolic enzymes in cells have suggested that metabolism may benefit from spatial compartmentalization. Recently, we demonstrated the formation of a metabolic complex by the enzymes involved in human glucose metabolism in cells; namely the glucosome. Importantly, we have noticed the formation of large-sized glucosome clusters in the cytoplasm of various human cancer cells, but not in non-cancerous, normal human cell lines. We hypothesize that the large-sized clusters of glucosomes are cancer-specific and thus functionally essential for cancer cell metabolism. To test this hypothesis, in Aim1, we will determine the cancer specificity of the large-sized clusters by analyzing the degree of the large-sized cluster formation and its impacts on metabolic profiles from various human cancer and non-cancerous cell lines. In Aim 2, we will detail size-dependent functional contribution of the glucosome clusters to cancer cell metabolism. We will carry out quantitative single-cell analyses with various fluorescence microscopic techniques and also 13C-metabolic flux analysis with mass spectrometry. The proposed research using both single-cell and ensemble measurements will uncover unprecedented mechanistic insights of the spatial alteration of glucose metabolism at single cancer cell levels. This new level of understanding will divulge the importance of a heretofore unrecognized metabolic complex, the glucosome, as a novel target for anti-cancer therapeutic intervention. Collectively, successful application of the proposed research will accelerate our efforts in translating our basic cancer research into novel drug discovery for the treatment of human cancer.